Mite allergen

ABSTRACT

A safe and efficient recombinant mite allergen is provided as a therapeutic agent or a diagnostic agent for mite allergic diseases, which contains no anaphylaxis-inducing impurities. The following recombinant protein (a) or (b) is provided:
     (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 or 35; or   (b) a protein comprising an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO: 2 or 35 by deletion, substitution, or addition of one or several amino acids and having mite allergen activity.

This application is a Continuation of U.S. patent application Ser. No.11/547,850, now U.S. Pat. No. 8,075,898 filed May 17, 2007, which is theNational Stage of International Application No. PCT/JP05/07191, filedApr. 7, 2005, which applications are considered as being a part of thisapplication, and the entire disclosures of application Ser. Nos.11/547,850 and PCT/JP05/07191 are expressly incorporated by referenceherein in their entireties. This application also claims priority toJapanese Patent Application No. JP 2004-116089, filed Apr. 9, 2004.

TECHNICAL FIELD

The present invention relates to recombinant mite allergens havingallergen activity and in particular relates to mite allergens that causeatopy in dogs. The present invention further relates to genes encodingthe allergens, expression vectors that enable expression of the genes,transformants obtained by transformation using expression vectors, amethod for producing the recombinant mite allergens, therapeutic agentsfor mite allergic diseases, and diagnostic agents for mite allergicdiseases.

BACKGROUND ART

House dust mites are known as major causes of allergic diseases such asatopic dermatitis and bronchial asthma. Conventionally, desensitizationtherapy that uses causative substances of allergies as therapeuticagents for allergic diseases is regarded as the most important basicremedy. In particular, the desensitization therapy is broadly conductedfor diseases such as pollinosis, house dust allergies, and fungalallergies, which are induced by antigens such as inhalant allergens thatare difficult to avoid. However, the desensitization therapy involvesthe risk of anaphylaxis due to the action of sensitizing antigens, sothat administration of safe therapeutic antigens is required. Such safesensitizing antigens are under research.

Regarding mite allergic diseases, 2 types of mites, Dermatophagoidespteronyssinus and Dermatophagoides farinae, have been reported asallergen sources in house dust (see Non-patent documents 1 and 2). Majormite allergens have been fractionated from these mites. These miteallergens are known to be a glycoprotein (pI 4.6 to 7.2) with amolecular weight between 24 kD and 28 kD and/or a protein (pI 5 to 7.2)with a molecular weight between 14.5 kD and 20 kD contained in miteexcretion and/or mite bodies (see Non-patent documents 3 to 7).

Regarding the mite allergen genes, Der p 1 (molecular weight: 25,371)and Der p 2 (molecular weight: 14,131) which are major allergens ofDermatophagoides pteronyssinus and Der f 1 (molecular weight: 25,191)and Der f 2 (molecular weight: 14,021) which are major allergens ofDermatophagoides farinae have been cloned and the nucleotide sequencesthereof have also been determined (see Non-patent documents 8 to 15).Recombinant allergens based on these allergens have also been prepared,and research concerning the same has proceeded. Moreover, the nucleotidesequence of Der f 3, an allergen with a molecular weight ofapproximately 30,000, has also been reported (see Non-patent document16). Furthermore, as mite allergens, ma 10, ma 3, ma 15, ma 29, ma 44,ma 50, ma 113, ma 114, and ma 115 (see Patent document 1) have also beenreported. Moreover, ma 124, which exerts strong crossreactivity with ananti-Der f 2 serum, has also been reported (see Patent document 2).

Furthermore, it has been reported concerning dogs that 98-kDa Der f 15,109-kDa Der f 15 (see Non-patent document 17), and 60-kDa Der f 18 (seeNon-patent document 18) are allergens with which IgE strongly reacts.

As a method for diagnosing mite allergic diseases, an intradermalreaction test has conventionally been used as a mainstream method, whichis based on a patient's history and uses house dust extracts and/or mitebody extracts. This method (test) has been used in combination with aRAST (radio allergosorbent test) method that involves serum IgE antibodytiter (relative value) measurement, an inhalation induction test, anasal mucous membrane provocation test, and the like. However, it hasremained considerably difficult to directly diagnose mite allergicdiseases.

A desensitization therapeutic method for bronchial asthma has beenconventionally performed, which uses a house dust extract and a housedust mite as a specific allergen. However, the composition of house dusthas not been analyzed sufficiently. Moreover, house dust contains manytypes of impurities that can induce anaphylaxis. Hence, the doses ofhouse dust in such cases are extremely limited. Accordingly,conventional desensitization treatment can have effects at extremely lowlevels. Therefore, more effective and safer antigens for desensitizationtreatment have been desired. It has been conventionally known thatallergens effective for desensitization treatment are present infractions of high-molecular-weight crude mite excretions. From suchfractions, it has been impossible to obtain mite allergens in amountssufficient for desensitization treatment. Therefore, with methods thatinvolve extraction and purification of mite allergens from productsobtained by raising mites, achieving a stable supply of antigens fortreatment is difficult. Furthermore, as described above, variousrecombinant mite allergens have been conventionally reported with theuse of gene recombination techniques. However, it cannot be said thatthese allergens are always effective for actual treatment. Provision ofa recombinant mite allergen with more effective, new, and greater miteallergen activity has been desired.

-   Patent document 1 JP Patent Publication (Kokai) No. 7-112999 A    (1995)-   Patent document 2 JP Patent Publication (Kokai) No. 7-278190 A    (1995)-   Non-patent document 1 Allerg. Asthma, 10, 329-334 (1964)-   Non-patent document 2 J. Allergy, 42, 14-28 (1968)-   Non-patent document 3 J. Immunol., 125, 587-592 (1980)-   Non-patent document 4 J. Allergy Clin. Immunol., 76, 753-761 (1985)-   Non-patent document 5 Immunol., 46, 679-687 (1982)-   Non-patent document 6 Int. Arch. Allergy Appl. Immunol., 81, 214-223    (1986)-   Non-patent document 7 J. Allergy Clin. Immunol., 75, 686-692 (1985)-   Non-patent document 8 Int. Arch. Allergy Appl. Immunol., 85, 127-129    (1988)-   Non-patent document 9 J. Exp. Med., 167, 175-182 (1988)-   Non-patent document 10 J. Exp. Med., 170, 1457-1462 (1989)-   Non-patent document 11 Int. Arch. Allergy Appl. Immunol., 91,    118-123 (1990)-   Non-patent document 12 Int. Arch. Allergy Appl. Immunol., 91,    124-129 (1990)-   Non-patent document 13 Jpn. J. Allergol., 39, 557-561 (1990)-   Non-patent document 14 Clinical and Experimental Allergy, 21, 25-32    (1991)-   Non-patent document 15 Clinical and Experimental Allergy, 21, 33-37    (1991)-   Non-patent document 16 FEBS Lett., 377, 62-66 (1995)-   Non-patent document 17 Vet. Immunol. Immunopathol, 78, 231-247    (2001)-   Non-patent document 18 J. Allergy Clin. Immunol, 112, 79-86 (2003)

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide safe and effectiverecombinant mite allergens containing no anaphylaxis-inducing impuritiesas therapeutic agents or diagnostic agents for mite allergic diseases.More specifically, objects of the present invention are to provide genesderived from mite bodies and to provide expression vectors that enableexpression of the genes. Still another object of the present inventionis to provide novel mite allergens having allergen activity, which areobtained by expression of genes derived from mite bodies. Furtherobjects of the present invention are to provide novel therapeutic agentsfor mite allergic diseases containing recombinant mite allergens asactive ingredients and to provide novel diagnostic agents for miteallergic diseases containing recombinant mite allergens.

As a result of intensive studies to achieve the above objects, thepresent inventors have discovered novel mite allergens and alsodiscovered that the allergens exert excellent effects in desensitizationtreatment. Hence, the present inventors have completed the presentinvention.

Specifically, the present invention is as described below.

[1] The following recombinant mite allergen (a) or (b):

(a) a recombinant mite allergen comprising the amino acid sequencerepresented by SEQ ID NO: 2 or 35; or

(b) a recombinant mite allergen comprising an amino acid sequencederived from the amino acid sequence represented by SEQ ID NO. 2 of 35by deletion, substitution, or addition of one or several amino acids andhaving mite allergen activity.

[2] A gene encoding the following mite allergen (a) or (b):

(a) a mite allergen comprising the amino acid sequence represented bySEQ ID NO: 2 or 35; or

(b) a mite allergen comprising an amino acid sequence derived from theamino acid sequence represented by SEQ ID NO: 2 or 35 by deletion,substitution, or addition of one or several amino acids and having miteallergen activity.

[3] A gene comprising the following DNA (c) or (d):

(c) a DNA comprising the nucleotide sequence represented by SEQ ID NO: 1or 34; or

(d) a DNA hybridizing under stringent conditions to a DNA comprising asequence complementary to that of the DNA comprising the nucleotidesequence of SEQ ID NO: 1 or 34 and encoding a protein having miteallergen activity.

[4] A fragment peptide of the mite allergen according to [1].

[5] The fragment peptide according to [4], which comprises an amino acidsequence that contains at least one of the amino acid sequencesrepresented by SEQ ID NO: 3 to SEQ ID NO: 19.

[6] A fragment gene of a mite allergen, which encodes the fragmentpeptide according to [4] or [5].

[7] A recombinant vector, which contains the gene according to [2] or[3] or the fragment gene according to [6].

[8] A fusion protein, which is composed of the mite allergen accordingto [1] and another protein.

[9] A bacterial, yeast, insect, or animal cell, which is transformedusing the expression vector according to [7].

[10] A method for producing a recombinant mite allergen, which comprisesculturing the bacterial, yeast, insect, or animal cell according to [9]under conditions in which the gene can be expressed, causing the cell toproduce a recombinant mite allergen, and then harvesting the recombinantmite allergen.[11] A method for producing a recombinant mite allergen, which comprisesculturing the bacterial, yeast, insect, or animal cell according to [9]under conditions in which the gene can be expressed, causing the cell toproduce a fusion recombinant mite allergen, harvesting the fusionrecombinant mite allergen, and then eliminating the other protein fusedto the allergen.[12] A therapeutic agent for mite allergic diseases, which contains asan active ingredient the recombinant mite allergen according to [1], thefragment peptide according to [4], or the fusion protein according to[8].[13] A diagnostic agent for mite allergic diseases, which contains as anactive ingredient the recombinant mite allergen according to [1], thefragment peptide according to [4], or the fusion protein according to[8].[14] An antibody against the mite allergen according to [1].[15] The antibody against the mite allergen according to [14], which isa monoclonal antibody.[16] A hybridoma, which produces the monoclonal antibody according to[15].[17] An immunoassay method for a mite allergen in house dust, which usesthe antibody according to any one of [14] to [16].[18] The immunoassay method for a mite allergen in house dust accordingto [17], which is the ELISA method.

This specification includes part or all of the contents as disclosed inthe description and/or drawings of Japanese Patent Application No.2004-116089, which is a priority document of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing the result of electrophoresis of arecombinant dog FcεRIα chain.

FIG. 2 is a graph showing the binding of a recombinant dog FcεRIα chainwith IgE.

FIG. 3 shows the result of detecting Dermatophagoides farinae-specificIgE.

FIG. 4 is a photograph showing the results of Western blot analysisusing a recombinant dog FcεRIα chain.

FIG. 5 is a photograph showing a 2-D (two-dimensional) electrophoresispattern of an antigen extracted from Dermatophagoides farinae.

FIG. 6 shows photographs showing the results of 2-D (two-dimensional)electrophoresis and Western blot analysis of a Dermatophagoides farinaeallergen protein.

FIG. 7-1 shows the partial nucleotide sequence (SEQ ID NO: 1) and aminoacid sequence (SEQ ID NO: 2) of a Zen1 gene.

FIG. 7-2 shows the partial nucleotide sequence and amino acid sequenceof the Zen1 gene (continuation of FIG. 7-1).

FIG. 8-1 shows the full-length cDNA nucleotide sequence (SEQ ID NO: 34)and amino acid sequence (SEQ ID NO: 35) of the Zen1 gene.

FIG. 8-2 shows the full-length cDNA nucleotide sequence and amino acidsequence of the Zen1 gene (continuation of FIG. 8-1).

FIG. 8-3 shows the full-length cDNA nucleotide sequence and amino acidsequence of the Zen1 gene (continuation of FIG. 8-2).

FIG. 8-4 shows the full-length cDNA nucleotide sequence and amino acidsequence of the Zen1 gene (continuation of FIG. 8-3).

FIG. 9 is a photograph showing the SDS-PAGE result of recombinant Zen1prepared using Escherichia coli and then purified.

FIG. 10 is a photograph showing the result of a reaction analyzed byWestern blotting of an anti-Zen 1 polyclonal antibody. Lane 1 shows theresult with regard to a recombinant Zen1 and Lane 2 shows the resultwith regard to a mite body.

FIG. 11 is a graph showing the reactivity of recombinant Zen1 with IgEas analyzed by ELISA.

BEST MODE OF CARRYING OUT THE INVENTION

The present invention will be described in detail as follows.

(1) Isolation of a Mite Allergen Zen1 Protein and Determination ofPartial Sequences Thereof

A mite allergy is specified using mite-allergen-specific IgE obtainedfrom an animal clinically diagnosed as having a mite allergy.Specifically, a mite allergen is identified by Western blotting usingserum that contains mite-allergen-specific IgE, a mite extract, and anIgE receptor that recognizes allergen-specific IgE, for example. Anallergen can be identified by a known method.

The thus identified novel mite allergen of the present invention, whichis a Zen1 protein, has a molecular weight between 150 kDa and 200 kDa.

The identified mite allergen can be isolated by performingelectrophoresis and then extracting the mite allergen from a miteallergen spot. At such time, it is desired to perform 2-D(two-dimensional) electrophoresis for complete separation from otherproteins.

Partial sequences can be determined by a known method using the thusextracted mite allergen. Examples of known methods for determiningpartial sequences include de novo sequencing based on MS/MS and peptidemapping.

(2) Preparation of cDNA Clone by RT-PCR

A DNA encoding the mite allergen of the present invention can beobtained by extracting mRNA from a mite, synthesizing a mite allergencDNA using the mRNA as a template, constructing a cDNA library, and thenscreening for the target.

A supply source of such mRNA is a mite body, and the mite is preferablyDermatophagoides farinae, Dermatophagoides pteronyssinus, or the like,which are house dust mites. However, the examples are not limitedthereto. Such mRNA can be prepared by generally employed techniques. Thethus obtained mRNA is used as a template, primers are designed based onthe sequence information obtained in (1) above, and then a cDNA fragmentencoding a mite allergen is synthesized. The thus obtained fragment issubcloned to an appropriate vector such as pGEM (produced by Promega).The nucleotide sequence is then determined by a standard method such asa cycle sequencing method.

Partial amino acid sequences of the mite allergen of the presentinvention are shown in SEQ ID NOS: 3 to 19. Of these, the sequencesshown in SEQ ID NOS: 3 to 7 were determined by de novo sequencing. AnN-terminal amino acid sequence is shown in SEQ ID NO: 19. The sequencesshown in SEQ ID NOS: 8 to 18 were determined by peptide mapping.

The present invention includes a mite allergen that comprises an aminoacid sequence comprising at least one of the amino acid sequences shownin SEQ ID NOS: 3 to 19 that represent fragments of the Zen1 protein,which is a mite allergen.

A partial nucleotide sequence of the DNA of the Zen1 gene encoding theZen1 protein, which is the mite allergen of the present invention, isshown in SEQ ID NO: 1, and the full-length nucleotide sequence thereofis shown in SEQ ID NO: 34. A partial amino acid sequence of the Zen1protein, which is the mite allergen of the present invention, is shownin SEQ ID NO: 2, and the full-length amino acid sequence thereof isshown in SEQ ID NO: 35.

As long as a protein comprising such amino acid sequence has miteallergen activity, mutations such as deletion, substitution, or additionof at least one, and preferably one or several, amino acid(s) may takeplace in the amino acid sequence.

For example, at leak one, and preferably one or several (e.g., 1 to 10and further preferably 1 to 5), amino acid(s) of the amino acid sequencerepresented by SEQ ID NO: 2 or SEQ ID NO: 35 may be deleted. At leastone, and preferably one or several (e.g., 1 to 10 and further preferably1 to 5), amino acid(s) may be added to the amino acid sequencerepresented by SEQ ID NO: 2. Alternatively, at least one, and preferablyone or several (e.g., 1 to 10 and further preferably 1 to 5), aminoacid(s) of the amino acid sequence represented by SEQ ID NO: 2 may besubstituted with (an)other amino acid(s).

Examples of such amino acid sequence derived from the amino acidsequence of SEQ ID NO: 2 or SEQ ID NO: 35 by deletion, substitution, oraddition of one or several amino acids include amino acid sequenceshaving at least 85% or more, preferably 90% or more, further preferably95% or more, and particularly preferably 97% or more homology with theamino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 35 as calculated usingBLAST (Basic Local Alignment Search Tool at the National Center forBiological Information) using default parameters for initial setting,for example.

A protein having such amino acid sequence derived from the amino acidsequence of SEQ ID NO: 2 or SEQ ID NO: 35 by deletion, substitution, oraddition of one or several amino acids is substantially the same as theprotein having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 35.

Furthermore, examples of the gene of the present invention also includea DNA that is capable of hybridizing under the following conditions to aDNA comprising a sequence complementary to that of a gene having the DNAsequence shown in the above SEQ ID NO: 1 or SEQ ID NO: 34 and thatencodes a protein having mite allergen activity. Specifically, suchconditions enable identification by hybridization in the presence of 0.7M to 1.0 M NaCl at 68° C. using a filter on which a DNA is immobilizedand washing with the use of a 0.1 to 2×SSC solution (1×SSC comprises 150mM NaCl and 15 mM sodium citrate) at 68° C. Alternatively, the gene ofthe present invention is a DNA that can form a hybrid when it istransferred to and immobilized on a nitrocellulose membrane by theSouthern blotting method and then allowed to react overnight at 42° C.in a hybridization buffer (50% formamide, 4×SSC, 50 mM HEPES (pH 7.0),10×Denhardt's solution, and 100 μg/ml salmon sperm DNA).

Furthermore, the present invention also includes an RNA corresponding tothe above DNA or an RNA capable of hybridizing under stringentconditions to the RNA and encoding a protein having mite allergenactivity.

The recombinant vector of the present invention can be obtained byligating (inserting) the gene of the present invention into anappropriate vector. Vectors for use in insertion of the gene of thepresent invention are not particularly limited as long as they arereplicable in hosts such as bacteria, yeast, or animal cells. Examplesof such vectors include a plasmid DNA and a phage DNA. A vector DNA thatis used for construction of an expression vector is widely disseminatedand easily obtained. Examples of such vector DNA include pUC19 andpTV118 N (produced by Takara Shuzo), pUEX2 (produced by Amersham),pGEX-4T and pKK233-2 (produced by Pharmacia), and pMAM-neo (produced byClontech).

A method for constructing such expression vector of the presentinvention is not particularly limited and can be performed according toa standard method. For example, a mite allergen cDNA fragment digestedwith EcoR I can be inserted into the EcoR I site in the plasmid pUC19multicloning site. Furthermore, the fragment can be ligated to the EcoRI site of a plasmid vector pGEX-4T, so that an expression vector can beobtained.

Bacteria, yeast, or animal cells transformed with such expression vectorof the present invention are not particularly limited, as long as theycan express the gene of the present invention. Examples of such bacteriainclude Escherichia coli and Bacillus subtilis. Examples of such yeastinclude Saccharomyces cerevisae and the like. Examples of such animalcells include Chinese hamster ovary (CHO) cells, Sf21 and Sf9 cellswhich are Mamestra brassicae ovarian cells, monkey COS cells, and mousefibroblasts.

Examples of the recombinant mite allergen of the present inventioninclude, in addition to mite allergens that are directly expressed,those expressed as fusion proteins with other proteins. Hereinafter,such fusion proteins are referred to as fusion recombinant miteallergens. Examples of other proteins that form such fusion proteinsinclude, but are not particularly limited to, β-galactosidase,glutathione S-transferase, protein A, and a maltose-binding protein.

The recombinant mite allergen of the present invention may also be apeptide fragment consisting of only a region essential for allergenactivity or a peptide fragment comprising a region essential forallergen activity. Moreover, in addition to a product obtained byexpression of a mite allergen protein alone, such recombinant miteallergen may be obtained from a product expressed as a fusion protein byeliminating the other protein(s).

Specifically, the recombinant mite allergen of the present invention isobtained by expression of a gene derived from a mite body and is aprotein having mite allergen activity. Here, “having mite allergenactivity” means to be capable of inducing an allergy reaction in amammal.

The mite allergen of the present invention can be produced by thefollowing methods. After completion of culture of the above transformantstrain, microbial bodies are harvested, suspended in a buffer containingvarious protease inhibitors, and then disrupted by ultrasonication. Amembrane-localized protein in cell debris is extracted using a buffercontaining a protease inhibitor such as phenylmethanesulfonyl fluoride,monoiodoacetic acid, pepstatin A, or ethylenediaminetetraacetic acid anda surfactant such as sodium lauryl sulfate (SDS), triton X-100, orNonidet P40. A fusion protein composed of the mite allergen andglutathione S-transferase obtained from the extract or the cultureconcentrate is purified by affinity chromatography using immobilizedglutathione, affinity chromatography using immobilized anti-mite bodyantibody, or the like. In addition, a carrier on which glutathione isimmobilized is a carrier produced by Pharmacia. Furthermore, a carrieron which an anti-mite body antibody is immobilized is a carrier preparedby covalently binding a rabbit anti-mite body antibody to an activatedTresyl carrier (e.g., Tresyl GM gel (produced by Kurita WaterIndustries), Tresyl Toyopearl (produced by Tosoh), and Tresyl sepharose(produced by Pharmacia)). Furthermore, a fusion protein composed of amite allergen and a His tag (e.g., 6×His) can be obtained, followed bypurification using affinity beads to which a metal is immobilized, orthe like.

The purified fusion recombinant mite allergen is digested with proteaseand then fractionated by a single or a combination of known purificationmethod(s) including gel filtration chromatography, ultrafiltration, ionexchange chromatography, affinity chromatography, hydrophobicchromatography, chromatofocusing, an isoelectric focusing method, and agel electrophoresis method while monitoring takes place with ELISA and aleukocyte histamine release test for mite allergic disease patients(Allergy 37, 725 (1988)).

The present invention also encompasses a therapeutic agent for miteallergic diseases containing a mite allergen as an active ingredient.Such therapeutic agent is used as a therapeutic agent for various typesof mite allergic diseases. Here, “mite allergic diseases” means allallergic diseases that are caused by mite-specific antigens, such asatopic bronchial asthma, allergic rhinitis, allergic conjunctivitis, andatopic dermatitis.

The therapeutic agent for mite allergic diseases of the presentinvention can be prepared by drying a recombinant mite allergen or afragment peptide thereof purified by the above method, harvesting suchallergen or fragment peptide in a powdery form, and then preparing atherapeutic desensitizing agent for mite allergic diseases, for example.However, the method is not particularly limited thereto. When thetherapeutic agent for mite allergic diseases of the present invention isused as a therapeutic desensitizing agent, the agent can be directlyused or, if necessary, used as a combination drug supplemented by astandard method with a generally used adjuvant and various additiveagents such as a stabilizing agent, an excipient, a solubilizing agent,an emulsifying agent, a buffer agent, a soothing agent, a preservative,and a coloring agent. For example, a purified recombinant mite allergenin a powdery form is dissolved in physiological saline supplemented withphenol and then used as a stock solution for an antigen fordesensitization treatment.

The therapeutic agent for mite allergic diseases of the presentinvention can be administered via general routes of administration suchas percutaneous, oral, intracutaneous, subcutaneous, intramuscular, andintraperitoneal administration methods. Furthermore, the therapeuticagent of the present invention can also be used in a percutaneous ortransmucosal drug such as a troche, a sublingual tablet, an eye drop, anintranasal spray agent, a poultice, a cream, and a lotion. Furthermore,the dose and the number of instances of administration of thetherapeutic agent for mite allergic diseases of the present inventionare appropriately selected depending on the route of administration,symptoms, and the like, so that the dose is within a range ofapproximately 20 μg or less per instance of administration for an adult.Administration is performed once or several times a week.

Furthermore, the therapeutic agent for mite allergic diseases of thepresent invention is useful not only as a therapeutic agent against miteallergic diseases, but also as a prophylactic agent against the same.Moreover, the therapeutic agent for mite allergic diseases of thepresent invention can be safely used for human bodies without theexertion of anaphylaxis-inducing action.

The diagnostic agent for mite allergic diseases of the present inventionis used as a reagent for diagnosing intracutaneous reactions againstmite allergic diseases or a titration reagent for diagnosing miteallergies. When the diagnostic agent is used as a reagent for diagnosingintracutaneous reactions, the reagent is obtained by preparing arecombinant mite allergen or a fragment peptide thereof purified by theabove method according to a standard method. For example, a recombinantmite allergen is dried and powdered, the powder is dissolved and dilutedin physiological saline containing phenol, and then it is used. A methodusing the diagnostic agent as a reagent for diagnosing intracutaneousreactions is employed according to a standard method.

Furthermore, when the diagnostic agent is used as a titration reagentfor diagnosing mite allergies, the reagent is prepared similarly by astandard method. For example, a recombinant mite allergen or a fragmentpeptide thereof is appropriately dissolved and diluted in a Hank'sbuffer, so that the resultant is used as a reagent for histamine releasetitration. This method is generally performed by the followingprocedures. Specifically, blood of a mite allergic disease patient or ablood cell fraction obtained by centrifugation from the blood of thepatient is suspended in a buffer. A fixed amount of the blood cellsuspension is subjected to titration using a recombinant mite allergenas a titration reagent. The amount of histamine that is released frombasophils by allergen stimulation is measured using HPLC [Allergy 37,725 (1988)].

In the histamine release titration, the amount of histamine to bereleased is determined based on 50% (inflexion point of the titrationcurve) of the maximum release amount. Specifically this titration ischaracterized in that: (1) a patient's allergen sensitivity is directlymeasured based on a titer of a blood cell suspension; and (2) afterpre-reaction of blood plasma with a recombinant mite allergen, the value(blood titration curve value) obtained by titrating the blood cellsuspension with the reaction solution is usually higher than the value(blood cell suspension titration curve value) obtained by titrating theblood cell suspension with the recombinant mite allergen. This is due tothe presence of an IgG antibody (blocking antibody) capable of allergenneutralization in blood plasma. Therefore, the blocking antibody titercan be obtained from the degree to which the blood titration curve hasshifted from the blood cell suspension titration curve. The allergensensitivity and this blocking antibody titer enable accurate miteallergy diagnosis feasible. This histamine release titration test isalso useful for monitoring the effect of desensitization treatment.

The present invention also encompasses an antibody against the miteallergen of the present invention or a fragment peptide thereof. Suchantibody can be obtained as a polyclonal antibody or a monoclonalantibody by a known method. Such antibody can be used for measuring thepresence, the absence, or the like of a mite allergen in house dust, forexample. Such measurement can be performed by a known immunologicalmethod such as ELISA. Upon such measurement, a protein is extracted fromhouse dust and then measured.

In addition, the recombinant mite allergen protein of the presentinvention is expressed. By conducting a test such as a specific IgEreaction test or an intracutaneous reaction test with a mite allergypatient dog using the thus expressed recombinant protein, the allergenprotein functions of the mite allergen protein of the present inventioncan be confirmed.

The present invention will be further described in the followingexamples. The examples are not intended to limit the scope of theinvention.

In addition, reagents used in each example were commercial reagentspurchased from Nacalai Tesque, Wako Pure Chemical Industries, Sigma,Difco, or the like, unless otherwise specified. Furthermore, reagentsfor genetic engineering, such as restriction enzymes were purchased fromTakara Shuzo, Toyobo, Invitrogen, or the like and then used according tothe manufacturers' instructions.

Example 1 Establishment of an IgE Detection System

The extracellular region of a dog high-affinity IgE receptor α chain(FcεRIα) cDNA, excluding its signal peptide site and having hadrestriction enzyme EcoR I and Xho I sites added thereto, was amplifiedby PCR. The resultant was ligated to the EcoR I and Xho I sites ofEscherichia coli expression plasmid vector pGEX4T-1 (produced byAmersham Biosciences) using T4-DNA ligase. An E. coli TOP10 strain(produced by Invitrogen) was transformed with the thus obtainedrecombinant plasmid. The transformed strain was cultured at 37° C.overnight in an LB medium containing ampicillin (100 μg/mL).Subsequently, a small amount of the strain was subcultured on a new LBmedium until OD at 600 nm reached 1.0. Next, IPTG(isopropyl-1-thio-β-D-galactoside) was added to a final concentration of1 mM. 3 hours later, cells were harvested and then washed once with PBS(pH 7.4). Cells harvested again were lysed by ultrasonication in PBS (pH7.4), insoluble fractions were removed by centrifugation, and thensoluble fractions containing dog FcεRIα fused to glutathioneS-transferase (GST) were collected. Subsequently, dog FcεRIα fused toGST was obtained from the soluble fractions using a glutathionesepharose 4B column (produced by Amersham Biosciences). 1.0 mg of thefusion protein (GST-FcεRIα) was obtained from 10 liters of the culturesolution. It was confirmed that the obtained purified GST-FcεRIα showeda single band of 45 kDa as a result of SDS-PAGE (FIG. 1). Recombinantdog IgE (produced by BETHYL) and purified dog IgG were immobilized on animmunoplate (produced by Nalge Nunc International) with 2-fold serialdilution from 1.0 μg, 0.1 μg, 0.05 μg, 0.025 μg, 0.0125 μg, and then0.00625 μg, so as to confirm the reactivity of the purified GST-FcεRIα.It was confirmed that the purified GST-FcεRIα reacted with IgE but didnot react with IgG (FIG. 2). 4.0 μg of an antigen extracted fromDermatophagoides farinae (produced by GREER) was immobilized on animmunoplate (produced by Nalge Nunc International).Dermatophagoides-farinae-positive dog serum (dog serum that testedpositive for Dermatophagoides farinae as confirmed by an intracutaneousreaction using a Dermatophagoides farinae antigen solution (produced byGREER) diluted 50-fold using physiological saline) was caused to reactwith the antigen. Biotin-labeled GST-FcεRIα was then added. Furthermore,based on a color development reaction resulting from the addition ofperoxidase conjugate streptavidin (produced by Jackson Immuno Research)and a substrate, it was confirmed that the GST-FcεRIα chain recognizedmite-allergen-specific IgE (FIG. 2). Furthermore, 4.0 μg of an antigenextracted from Dermatophagoides farinae (produced by GREER) wasimmobilized on an immunoplate (produced by Nalge Nunc International).Dog serum obtained by immunization with the antigen extracted fromDermatophagoides farinae was caused to react with the antigen. It wasconfirmed that the GST-FcεRIα reacted with neitherDermatophagoides-farinae-positive dog serum subjected, to heat treatmentat 56° C. for 1 hour nor dog purified IgG, but reacted with therecombinant dog IgE (produced by BETHYL). It was thought that theGST-FcεRIα recognized allergen-specific IgE (FIG. 3). In FIG. 3, “−”denotes serum not treated at 56° C. for 1 hour, “+” denotes serumtreated at 56° C. for 1 hour, and “cont.” denotes non-immunized serum.

Example 2 Analysis of a Major Mite Allergen by Western Blotting

Western blot analysis was conducted using the sera and blood plasmasamples of eight dogs. The eight dogs had developed atopic dermatitisand had been diagnosed with mite allergies based on intracutaneousreactions using the solution of an antigen extracted from aDermatophagoides farinae allergen (produced by GREER) and the ELISAmethod using the recombinant dog FcεRIα chain established in Example 1.β-mercaptoethanol was added to 100.0 μL of the solution of an antigenextracted from Dermatophagoides farinae to a final concentration of 50.0μL/mL. 200 μL of the thus prepared Laemmli sample buffer (produced byBIO-RAD) was added, followed by heat treatment at 100° C. for 5 minutes.The resultant was applied to polyacrylamide gel (PAGEL; produced byATTO) with a gel concentration between 5% and 20%, and thenelectrophoresis was performed. After completion of electrophoresis, theresultant was transferred to a PVDF membrane (Hybond-P; produced byAmersham Biosciences). The membrane was allowed to stand at 4° C.overnight in a blocking solution (PBST (prepared by adding Tween20 toPBS to a final concentration of 0.1%) supplemented with 5% skim milk).After the membrane had been washed in PBST for 10 minutes, each dogserum or blood plasma sample was diluted 10-fold in a blocking solution.Each diluted solution sample was added to the membrane, followed by 3hours of reaction at room temperature. Three instances of washing (10minutes each) were performed with PBST. Biotin-labeled dog FcεRIα wasdiluted with a blocking solution and then the diluted solution was addedto the membrane, followed by 2 hours of reaction at room temperature.After 3 instances of washing (10 minutes each) with PBST, astreptavidin-HRP conjugate (produced by Amersham Biosciences) diluted10,000-fold with PBST was added to the membrane, followed by 1 hour ofreaction at room temperature. After 5 instances of washing (10 minuteseach) with PBST, a reaction solution for an ECL Plus Western blottingdetection system (produced by Amersham Biosciences) was added onto themembrane, followed by 5 minutes of reaction at room temperature. Signalswere then detected using X-ray film (Hyperfilm ECL; produced by AmershamBiosciences). As a result, a protein showing a strong reaction wasdetected between a band corresponding to a molecular weight of 150 kDaand a band corresponding to the same of 250 kDa (FIG. 4). In FIG. 4, aband indicated with an arrow corresponds to the protein showing a strongreaction and having a molecular weight between 150 kDa and 250 kDa. InFIG. 4, numerals from 1 to 8 separately denote eight dogs, and “ct.”denotes a negative serum.

Example 3 Analysis of a Mite Allergen Protein by 2-D (Two-Dimensional)Electrophoresis

An allergen protein with a molecular weight between 150 kDa and 250 kDa,with which IgE had strongly reacted, was isolated by 2-D(two-dimensional) electrophoresis. 2-D (two-dimensional) electrophoresiswas performed using a Protean IEF cell (produced by BIO-RAD) 1.0 mg ofan antigen extracted from Dermatophagoides farinae (produced by GREER)was dissolved in 1.0 mL of a swelling buffer (2-D starter kit; producedby BIO-RAD). 300 μL of the thus obtained solution was swollen underactive conditions (50 V, 20° C., and 12 hours) using 17-cm-long IPGReady strip gel (pH 4-7, produced by BIO-RAD) and a focusing tray. Afterswelling, focusing was performed under the following conditions. First,a procedure to remove excessive salts was performed in step 1 (250 V, 20minutes, and 20° C.). In step 2, voltage was elevated from 250 V to10,000 V for 6 hours. In step 3, focusing was performed with a voltageof 10,000 V and with voltage hours totaling 60,000 VH. Before 2-D(two-dimensional) electrophoresis, the IPG ready strip gel was gentlyshaken for 10 minutes using SDS-PAGE equilibrated buffer I (6 M urea,0.375 M Tris pH 8.8, 2% SDS, 20% glycerol, and 2% (w/v) DTT; produced byBIO-RAD). Subsequently, the gel was further gently shaken for 10 minutesusing SDS-PAGE equilibrated buffer II (6 M urea, 0.375 M Tris pH 8.8, 2%SDS, 20% glycerol, and 2.5% (w/v) iodoacetamide; produced by BIO-RAD),thereby performing equilibration. The equilibrated IPG ready strip gelwas caused to closely adhere to PII ready gel (8-16%; produced byBIO-RAD) using 1% (v/w) low melt agarose (produced by BIO-RAD).Electrophoresis was performed with a 40-mA constant current (withinitial voltage of 135 V and final voltage of 400 V) for approximately 3hours. After electrophoresis, the gel was stained using Bio-Safe(produced by BIO-RAD), so that pattern analysis could be conducted forthe protein spot (FIG. 5).

Example 4 Identification of an Allergen Spot by Western Blot Analysis

After 2-D (two-dimensional) electrophoresis, the protein spot wastransferred to a PVDF membrane (Hybond-P; produced by AmershamBiosciences). The membrane was allowed to stand at 4° C. overnight in ablocking solution (PBST (prepared by adding Tween20 to PBS to a finalconcentration of 0.1%) supplemented with 5% skim milk). After themembrane had been washed with PBST for 10 minutes, a dog serum or bloodplasma sample was diluted 10-fold using a blocking solution. The thusdiluted solution was added to the membrane, followed by 3 hours ofreaction at room temperature. After 3 instances of washing (10 minuteseach) with PBST, biotin-labeled dog FcεRIα was diluted with a blockingsolution. The diluted biotin-labeled dog FcεRIα was added to themembrane, followed by 2 hours of reaction at room temperature. After 3instances of washing (10 minutes each) with PBST, a streptavidin-HRPconjugate (produced by Amersham Biosciences) diluted 10,000 fold withPBST was added to the membrane. One hour of reaction was performed atroom temperature. After 5 instances of washing (10 minutes each) withPBST, a reaction solution for an ECL plus Western blotting detectionsystem (produced by Amersham Biosciences) was added onto the membrane,followed by 5 minutes of reaction at room temperature. Signals weredetected using X-ray film (Hyperfilm ECL; produced by AmershamBiosciences). As a result, a spot showing a strong reaction was detectedbetween a band corresponding to a molecular weight of 150 kDa and a bandcorresponding to the same of 250 kDa and at a pH of about 4.5. Thus, thespot corresponding to the allergen protein (Zen1) of the presentinvention was obtained (FIG. 6). In FIG. 6: upper left FIG. 6-1 showsthe 2-D (two-dimensional) electrophoresis pattern (pH 4 to 7) ofDermatophagoides farinae; upper right FIG. 6-2 shows the result ofWestern blot analysis (pH 4 to 7) using theDermatophagoides-farinae-positive serum of a dog patient developingatopic dermatitis; lower left FIG. 6-3 shows the 2-D (two-dimensional)electrophoresis pattern (pH 3.9 to 5.1) of Dermatophagoides farinae; andlower right FIG. 6-4 shows the reaction spot (where the spot appears asa black circular spot on the upper portion in the figure and isindicated with an arrow) obtained by Western blot analysis (pH 3.9 to5.1) using the Dermatophagoides farinae-positive serum of a dog patientdeveloping atopic dermatitis, as compared with the 2-D (two-dimensional)electrophoresis pattern. A strong reaction was observed for the spotindicated with the arrow.

Example 5 Proteome Analysis of the Zen1 Protein

The Zen1 protein spot isolated by 2-D (two-dimensional) electrophoresiswas excised from the gel and then MS/MS analysis was conducted. A greatdeal of MS/MS data could be obtained, but no hits were confirmed. Fivepeptides thought to be novel proteins were subjected to the de novosequencing method, so that amino acid sequences (SEQ ID NOS: 3 to 7)were determined (Table 1). BLAST search was performed for these aminoacid sequences, but no clear hits were obtained.

TABLE 1 Partial sequence 415 MKSLLNEANELLK Partial sequence 445 SAQDVLEKPartial sequence 847 FMQSLLNEADELLR Partial sequence 448 LPDSDLKDELAKPartial sequence 491 LPDSDLKNELAEK Partial amino acid sequences of Zen1as determined by the de novo sequencing method.

Example 6 Peptide Mapping Analysis of the Zen1 Protein

The Zen1 protein spot isolated by 2-D (two-dimensional) electrophoresiswas excised from the gel. A peptide map was prepared and then amino acidsequencing was performed for 8 peaks. As a result, sequences (SEQ IDNOS: 8 to 18) of 11 amino acid fragments were determined (Table 2).BLAST search was performed, but no clear hits were obtained.

TABLE 2 Partial sequence 21 MYNFHLEAY Partial sequence 28 IAHFLELEPartial sequence 32 IAHFELE Partial sequence 23-1 KFQSLLNEAN Partialsequence 23-2 IAHLESE(T) Partial sequence 24 KFQSLLN(E)A Partialsequence 22 DAQLEXE Partial sequence 9-1 SAQDVSL Partial sequence 9-2RNEMNE Partial sequence 20-1 MFQSLLNKADFD Partial sequence 20-2 DLARDVXLAmino acid sequences corresponding to peaks obtained by peptide mappingof Zen1

Example 7 Analysis of the N-Terminal Amino Acid Sequence of the Zen1Protein

The Zen1 protein spot isolated by the 2-D (two-dimensional)electrophoresis was excised from the gel. The N-terminal amino acidsequence (SEQ ID NO: 19) of the Zen1 protein was determined by astandard method using an HP G1005A protein sequencing system (Table 3).BLAST search was performed for the sequence, but no clear hits wereobtained.

TABLE 3 N-terminal sequence DNRDDVLKQTEE Zen1 N-terminal amino acidsequence

Example 8 Extraction of Mite Total RNA and Separation of Mite Poly(A)mRNA

Untreated mite bodies obtained by culturing and growing Dermatophagoidesfarinae according to a standard method were placed in approximately 2.0L of a saturated saline solution. The solution was agitated well andthen allowed to stand for 30 minutes. Mite bodies in the supernatantwere skimmed using a strainer, washed using physiological saline, andthen dried 1.0 g of mite bodies was subjected to total RNA extractionand mite poly(A) mRNA separation using a PastTrack 2.0 kit (produced byInvitrogen) according to the manual of the kit.

Example 9 Synthesis of Mite cDNA

Reverse transcription reaction was performed using 100 ng of the mitepoly(A) mRNA separated in Example 8 as a template and a cDNA synthesiskit (ReverTraAce-α-; produced by Toyobo) according to the manual of thekit.

Example 10 Amplification of the Zen1 Gene by PCR

Based on the N-terminal amino acid sequence of the Zen1 protein, primersN-1 (5′-GAYGAYGTNTTRAARCARACNGARGAR-3′ (SEQ ID NO: 20): Y=C or T, N=A orC or G or T, and R=A or G) and N-2 (5′-GAY GAY GTN CTN AAR CAR ACN GARGAR-3′ (SEQ ID NO: 21): Y=C or T, N=A or C or G or T, and R=A or G) weredesigned as sense primers. Furthermore, 12 primers (SEQ ID NOS: 22 to33) were designed based on the amino acid sequences obtained by the denovo sequencing method (Table 4) as reverse primers. With the use of 1.0μg of the mite cDNA synthesized in Example 9 as a template, Ex taqpolymerase (produced by TaKaRa Bio), and each sample prepared accordingto the manual, thermal denaturation treatment was performed at 94° C.for 2 minutes and 35 cycles of reaction, each of which consisted of 94°C. for 1 minute, 65° C. for 2 minutes, and 72° C. for 3 minutes wereperformed. After further reaction was performed at 72° C. for 9 minutes,the sample was stored at 4° C. A DNA fragment of approximately 1,000 bpwas obtained when PCR was performed using a reverse primer 415-4(5′-RTTNAGNAGRTCYTTNGCRTCYTT-3′ (SEQ ID NO: 25): N=A or C or G or T, R=Aor G, and Y=C or T). A DNA fragment of approximately 880 bp was obtainedwhen PCR was performed using 491-2 (5′-RTT RTC NGC NAG RTC YTT RTT-3(SEQ ID NO: 29): N=A or C or G or T, R=A or G, and Y=C or T).

TABLE 4 N-1 5′- GAY GAY GTN TTR AAR CAR ACN GAR  GAR -3′ N-25′- GAY GAY GTN CTN AAR CAR ACN GAR  GAR -3′ 415-15′- RTT RAA RAA RTC YTT NGC RTC YTT  RAA -3′ 415-25′- RTT NAG RAA RTC YTT NGC RTC YTT -3′ 415-35′- RTT RAA NAG RTC YTT NGC RTC YTT -3′ 415-45′- RTT NAG NAG RTC YTT NGC RTC YTT -3′ 445-15′- RTT RTC RAA NAC YTC RTG NGC -3′ 445-25′- RTT RTC NAG NAC YTC RTG NGC -3′ 491-15′- RTT RTC NGC RAA RTC YTT RTT -3′ 491-25′- RTT RTC NGC NAG RTC YTT RTT -3′ 448-15′- RTT NGC RAA RTC YTC RTT RAA YTC -3′ 448-25′- RTT NGC NAG RTC YTC RTT RAA YTC -3′ 448-35′- RTT NGC RAA RTC YTC RTT NAG YTC -3′ 448-45′- RTT NGC NAG RTC YTC RTT NAG YTC -3′ Mixture primer sequencessynthesized for amplification of the Zen1 gene. A fragment ofapproximately 1000 bp was obtained when PCR was performed using N-1 and415-4. A fragment of approximately 880 bp was obtained when PCR wasperformed using N-1 and 491-2. N = A or C or G or T, R = A or G, Y = Cor T

Example 11 Cloning of the Zen1 Gene

The DNA fragment amplified using the primers N-1 and 415-4 in Example 10was collected from the agarose gel (SUPREC-01 produced by TaKaRa). TheDNA fragment was ligated to the cloning site of pGEM-T Easy Vector(produced by Promega) using T4 DNA ligase, thereby transforming hostEscherichia coli TOP10 (produced by Invitrogen). Specifically,Escherichia coli competent cells and a plasmid were mixed and then themixture was subjected to temperature treatment on ice for 30 minutes, at42° C. for 30 seconds, and on ice for 2 minutes. The resultant was thensuspended in an SOC medium (2% Trypton, 0.5% yeast extract, 0.05% NaCl,10 mM MgCl₂, 10 mM MgSO₄, and 20 mM glucose), followed by 1 hour ofincubation at 37° C. Subsequently, the transformed Escherichia coli wascultured at 37° C. overnight on an LB agar medium (1% yeast extract,0.5% trypton, and 1% NaCl) supplemented with 50 μg/ml ampicillin,thereby obtaining Escherichia coli colonies. White clones thought tocontain the inserted fragment were selected. The clones were culturedovernight on an LB medium supplemented with 50 μg/ml ampicillin. Theplasmid DNA was purified using a GFX (Trademark) Micro Plasmid Prep Kit(produced by Amersham Bioscience). Sequencing reaction was performedusing a dye primer cycle sequencing kit (produced by Amersham) and thennucleotide sequence analysis was performed using a fluorescence DNAsequencer (produced by Shimadzu Corporation). In addition, finaldetermination was made when the nucleotide sequences of 3 clones werefound to match completely upon the nucleotide sequence analysis thereof(FIG. 7-1 and FIG. 7-2).

Example 12 Analysis of the Zen1 Gene

The Zen1 gene cloned in Example 11 was analyzed using Genetyx-win ver.6software (produced by Software Development). The number of bases was1020 bp and the number of amino acid residues was 340 (FIG. 7-1, FIG.7-2, and SEQ ID NOS: 1 and 2). BLAST search was performed for thenucleotide sequence and the amino acid sequence of the gene, but noclear hits were obtained. The Zen1 gene was thought to be a novel gene.

Example 13 Isolation of Full-Length Zen1 cDNA

Full-length cDNA was isolated by the RACE (rapid amplification of cDNAends) method. Total RNA was extracted from the mite bodies used inExample 8 using an SV Total RNA isolation kit (produced by Promega). Atemplate for RACE was prepared using a GeneRacer (Trademark) kit(produced by Invitrogen) according to the manual of the kit.Furthermore, based on the Zen1 partial sequences obtained in Example 12,primers for 1^(st) PCR and nested PCR were synthesized for amplificationof the 5′ and the 3′ termini (Table 5). Amplification reaction for the5′ and the 3′ termini was performed as follows. To 1.0 μL of thetemplate for RACE prepared above, 3.0 μL each of 5′ and the 3′ primersincluded in a GeneRacer (Trademark) kit, 1.0 μL of a dNTP mix solution(10 mM each), 5.0 μl of 10×cDNA PCR reaction buffer included inAdvantage cDNA Polymerase Mix (produced by CLONTECH), 1.0 μL ofAdvantage cDNA Polymerase Mix, and 1.0 μL each of the gene-specificprimers for the 1^(st) PCR synthesized above and adjusted at 10 μM wereadded. Then the volume of the resulting solution was adjusted to 50.0 μLusing sterilized distilled water. Gene amplification was performed usinga Touchdown PCR method. The prepared sample solution was subjected tothermal denaturation at 94° C. for 1 minute, 5 cycles of reaction, eachof which consisted of 94° C. for 30 seconds and 72° C. for 4 minutes, 5cycles of reaction, each of which consisted of 94° C. for 30 seconds and70° C. for 4 minutes, 25 cycles of final reaction, each of whichconsisted of 94° C. for 30 seconds and 68° C. for 4 minutes, and thenstorage at 4° C. After completion of the 1^(st) PCR, to 1.0 mL each ofthe 5′ and the 3′ terminus amplification reaction solutions, 1.0 μL eachof 5′ primer and 3′ primer for Nested PCR included in a GeneRacer(Trademark) kit, 1.0 μL of a dNTP mix solution (10 mM each), 5.0 μl of10×cDNA PCR reaction buffer included in Advantage cDNA Polymerase Mix(produced by CLONTECH), 1.0 μL of Advantage cDNA Polymerase Mix, and 1.0μL each of the gene-specific primers for the 1st PCR synthesized aboveand adjusted at 10 μM were added. Then the volume of the resultingsolution was adjusted to 50.0 μL using sterilized distilled water. Geneamplification was performed using the above Touchdown PCR method. Thethus obtained amplified fragments of the 5′ and 3′ termini wereconfirmed by electrophoresis using 1.0% agarose gel. These fragmentswere excised and then collected (SUPREC-01 produced by TaKaRa). Thefragments were ligated to the cloning site of pGEM-T Easy Vector(produced by Promega) using T4 DNA ligase, thereby transforming hostEscherichia coli TOP10 (produced by Invitrogen). Specifically,Escherichia coli competent cells and a plasmid were mixed and then themixture was subjected to temperature treatment on ice for 30 minutes, at42° C. for 30 seconds, and on ice for 2 minutes. The resultant was thensuspended in an SOC medium (2% Trypton, 0.5% yeast extract, 0.05% NaCl,10 mM MgCl₂, 10 mM MgSO₄, and 20 mM Glucose), followed by 1 hour ofincubation at 37° C. Subsequently, the transformed Escherichia coli wascultured at 37° C. overnight on an LB agar medium (1% yeast extract, 0.5trypton, and 1% NaCl) supplemented with 50 μg/ml ampicillin, therebyobtaining Escherichia coli colonies. White clones thought to contain theinserted fragments were selected. The clones were cultured overnight onan LB medium supplemented with 50 μg/ml ampicillin. The plasmid DNA waspurified using a GFX (Trademark) Micro Plasmid Prep kit (produced byAmersham Bioscience). Sequencing reaction was performed using a dyeprimer cycle sequencing kit (produced by Amersham) and then nucleotidesequence analysis was performed using a fluorescence DNA sequencer(produced by Shimadzu Corporation). In addition, final determination wasmade when the nucleotide sequences of 3 clones were found to matchcompletely upon analysis thereof. As a result, the nucleotide sequenceof the 5′ terminus and that of the 3′ terminus of Zen1 could bedetermined (FIG. 8-1 to FIG. 8-4 and SEQ ID NOS: 34 and 35).

TABLE 5 Primer name Sequence Purpose of use Zen1 RS-15′- AAT TAC AAA CAT GAG TTA GAA -3′ 3′ RACE 1^(st) PCR Zen1 RS-25′- GAA TTG TTG ACA ATG TTC AAA -3′ 3′ RACE Nested PCR Zen1 RR-15′- GAT TTC ATC TTT CAA ATC TGA -3′ 5′ RACE 1^(st) PCR Zen1 RR-25′- CTT TTC CAA TAC ATC CTG GGC -3′ 5′ RACE Nested PCR (From the top,SEQ ID NOS: 36, 37, 38, and 39) Primers used in the RACE method and thesequences thereof

Example 14 Purification of Recombinant Zen1

Zen1 cDNA obtained in Example 13 to which restriction enzyme BamHI andXhoI sites had been added was amplified by PCR. The amplificationproduct was ligated to the BamHI and XhoI sites of the Escherichia coliexpression plasmid vector pGEX4T-1 (produced by Amersham Biosciences)using T4-DNA ligase. The E. coli TOP10 strain (produced by Invitrogen)was transformed with the thus obtained recombinant plasmid. Thetransformed strain was cultured at 37° C. overnight on an LB mediumcontaining 100 μg/mL ampicillin. A small amount of the strain wassubcultured on a new LB medium until OD at 600 nm reached 1.0. Next,IPTG (isopropyl-1-thio-β-D-galactoside) was added to a finalconcentration of 1 mM. 3 hours later, cells were harvested and thenwashed once with PBS (pH 7.4). Cells harvested again were lysed byultrasonication in PBS (pH 7.4), an insoluble fraction was removed bycentrifugation, and then a soluble fraction containing Zen1 fused toglutathione S-transferase (GST) was collected. Subsequently, Zen1 fusedto GST was obtained from the soluble fraction using a glutathionesepharose 4B column (produced by Amersham Biosciences). Thrombin(produced by Amersham Biosciences) in an amount 1/100 that of the fusionprotein was added to a solution containing a purified fusion product(that is, Zen1 fused to GST), followed by 20 hours of reaction at 22° C.Thus, GST was separated from Zen1. Subsequently, thrombin was removedusing Benzamidin Sepharose (produced by Amersham Biosciences) and then arecombinant Zen1 was purified. The thus obtained purified Zen1 showed asingle band corresponding to a molecular weight of approximately 60 kDaas confirmed by SDS-PAGE (FIG. 9).

Example 15 Preparation of an Anti-Zen1 Polyclonal Antibody and Analysisof the Reactivity of the Antibody with the Mite Protein

Six mice (BALB/c, female, 4-week-old) were immunized 5 times with therecombinant Zen1 purified in Example 14 at 1-week intervals.Subsequently, blood was collected from the mice, the sera wereseparated, and then the reaction of the IgG antibody was analyzed byELISA. ELISA was performed as follows. 1.0 μg of the recombinant Zen1and 4.0 μg of an antigen extracted from Dermatophagoides farinae(produced by GREER) were separately immobilized on immunoplates(produced by Nalge Nunc International), followed by 1 hour of blockingat 37° C. using a blocking solution (prepared by adding Tween20 to PBSsupplemented with 10% FBS to a final concentration of 0.05%). Each mouseserum sample was diluted 1000-fold with a blocking solution and thenallowed to react at room temperature for 1 hour. After washing eachimmunoplate, an HRP-labeled goat anti-mouse IgG monoclonal antibody(produced by ZYMED) diluted 2000-fold with a blocking solution wascaused to react therewith. After washing each immunoplate, 100 μL of anenzyme substrate solution (ABTS solution) was added to each well,followed by 10 minutes of reaction at 37° C. The enzyme reaction wasstopped by addition of 100 μL of a 0.32% sodium fluoride solution toeach well. The absorbance of each well at 414 nm was measured using animmunoreader (BioRad). After the reaction of IgG to the relevant subjectwas confirmed by ELISA, the subject was determined to be a polyclonalantibody against Zen1.

The reactivity of mouse IgG to the polyclonal antibody (the recombinantZen1) and the same with regard to mite bodies were analyzed by Westernblotting. 200 μL of a Laemmli sample buffer (produced by BIO-RAD) wasprepared by adding β-mercaptoethanol to a final concentration of 50.0μL/mL to 100.0 μL of the recombinant Zen1 protein solution adjusted at50 μg/mL and 100.0 μL of the solution of an antigen extracted fromDermatophagoides farinae. After heat treatment at 100° C. for 5 minutes,the resultant was applied to polyacrylamide gel (PAGEL; produced byATTO) with a gel concentration between 5% and 20%. Thus, electrophoresiswas performed. After completion of electrophoresis, the resultant wastransferred to a PVDF membrane (Hybond-P; produced by AmershamBiosciences). The membrane was allowed to stand at 4° C. overnight in ablocking solution (PBST (prepared by adding Tween20 to PBS to a finalconcentration of 0.1%) supplemented with 5% skim milk). The membrane waswashed with PBST for 10 minutes. An HRP-labeled goat anti-mouse IgGmonoclonal antibody (produced by ZYMED) was diluted 20,000-fold withPBST, and then the diluted solution was added onto the membrane,followed by 2 hours of reaction at room temperature. After 5 instances(10 minutes each) of washing with PBST, the reaction solution of an ECLPlus Western blotting detection system (produced by AmershamBiosciences) was added onto the membrane, followed by 5 minutes ofreaction at room temperature. Signals were detected using X-ray film(Hyperfilm ECL; produced by Amersham Biosciences). As a result, a signalindicating the reaction to the recombinant Zen1 with a molecular weightof approximately 60 kDa and a signal indicating the reaction to thenatural-type Zen1 with a molecular weight between 150 kDa and 250 kDawere detected (FIG. 10). Accordingly, it was confirmed that thefull-length Zen1 cDNA isolated in Example 13 encodes such allergenprotein of mite bodies with a molecular weight between 150 kDa and 250kDa.

Example 16 Analysis of IgE Reactivity of the Recombinant Zen1

The allergenicity of the recombinant Zen1 purified in Example 14 wasevaluated by ELISA. 1.0 μg of the recombinant Zen1 was immobilized on animmunoplate (produced by Nalge Nunc International K.K.), followed byblocking at 37° C. for 1 hour with a blocking solution (prepared byadding Tween20 to PBS supplemented with 10% FBS to a final concentrationof 0.05%). The sera of nine dogs that tested positive forDermatophagoides farinae (dog sera that tested positive forDermatophagoides farinae as confirmed by an intracutaneous reactionusing a Dermatophagoides farinae antigen solution (produced by GREER)diluted 50 fold using physiological saline)) were caused to react with anegative control dog serum. Biotin-labeled GST-FcεRIα was added and thena color development reaction was caused to take place by the addition ofperoxidase conjugate streptavidin (produced by Jackson Immuno Research)and an enzyme substrate solution (ABTS solution). The enzyme reactionwas stopped by the addition of 100 μL of a 0.32% sodium fluoridesolution per well. The absorbance of each well at 414 nm was measuredusing an immunoreader (BioRad). Specifically, the reaction of serum IgE(of nine dogs that had tested positive for mites as confirmed by theintracutaneous reaction of this procedure and of the negative dog(control)) to the recombinant Zen1 was analyzed by an ELISA system usingrecombinant dog FcεRIα. As a result, values above the value for thenegative dog (indicated with a dotted line) were confirmed. Therefore,it was confirmed that the recombinant Zen1 is an allergen protein thatreacts with IgE (FIG. 11).

INDUSTRIAL APPLICABILITY

The present invention makes it possible to provide safe and efficientrecombinant mite allergens as therapeutic agents or diagnostic agentsfor mite allergic diseases, which contain no anaphylaxis-inducingimpurities.

All publications cited herein are incorporated herein in their entirety.A person skilled in the art would easily understand that variousmodifications and changes of the present invention are feasible withinthe technical idea and the scope of the invention as disclosed in theattached claims. The present invention is intended to include suchmodifications and changes.

What is claimed is:
 1. A polypeptide fragment consisting of any one ofSEQ ID NOs: 2, 4, 10, 12, or
 19. 2. A therapeutic agent for miteallergic diseases, comprising a composition having the polypeptidefragment according to claim 1 as an active ingredient.
 3. A diagnosticagent for mite allergic diseases, wherein the diagnostic agent comprisesthe polypeptide fragment according to claim
 1. 4. A polypeptide fragmentof SEQ ID NO: 35 comprising any one of SEQ ID NOs: 4, 10, or
 19. 5. Apolypeptide fragment of SEQ ID NO: 35 comprising SEQ ID NO: 12 whereinthe amino acid at position 7 of SEQ ID NO: 12 is glutamic acid.